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Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival.

Vaillant AR, Mazzoni I, Tudan C, Boudreau M, Kaplan DR, Miller FD - J. Cell Biol. (1999)

Bottom Line: This convergent PI3-kinase-Akt pathway was essential for synergistic survival.In contrast, inhibition of calcium/calmodulin-dependent protein kinase II revealed that, while this molecule was essential for depolarization-induced survival, it had no role in KCl- induced Akt phosphorylation, nor was it important for synergistic survival by NGF and KCl.This convergent regulation of Akt may provide a general mechanism for coordinating the effects of growth factors and neural activity on neuronal survival throughout the nervous system.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4.

ABSTRACT
In this report, we have examined the mechanisms whereby neurotrophins and neural activity coordinately regulate neuronal survival, focussing on sympathetic neurons, which require target-derived NGF and neural activity for survival during development. When sympathetic neurons were maintained in suboptimal concentrations of NGF, coincident depolarization with concentrations of KCl that on their own had no survival effect, synergistically enhanced survival. Biochemical analysis revealed that depolarization was sufficient to activate a Ras-phosphatidylinositol 3-kinase-Akt pathway (Ras-PI3-kinase-Akt), and function-blocking experiments using recombinant adenovirus indicated that this pathway was essential for approximately 50% of depolarization-mediated neuronal survival. At concentrations of NGF and KCl that promoted synergistic survival, these two stimuli converged to promote increased PI3-kinase-dependent Akt phosphorylation. This convergent PI3-kinase-Akt pathway was essential for synergistic survival. In contrast, inhibition of calcium/calmodulin-dependent protein kinase II revealed that, while this molecule was essential for depolarization-induced survival, it had no role in KCl- induced Akt phosphorylation, nor was it important for synergistic survival by NGF and KCl. Thus, NGF and depolarization together mediate survival of sympathetic neurons via intracellular convergence on a Ras-PI3-kinase-Akt pathway. This convergent regulation of Akt may provide a general mechanism for coordinating the effects of growth factors and neural activity on neuronal survival throughout the nervous system.

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The role of CaMKII in survival mediated by NGF and/or KCl. a and b, CaMKII is necessary for KCl-mediated survival, but plays no role in the synergistic survival seen with NGF plus KCl. NGF-selected sympathetic neurons maintained in NGF and/or KCl for two days with or without 10 μM KN-62 and survival was then monitored by MTT assay (a) or TUNEL (b). In a, results represent the mean ± SD of one representative experiment performed in triplicate. Similar results were obtained in three separate experiments. In c, results represent the mean percentage of TUNEL-negative cells/total Hoechst-positive nuclei as determined in three separate experiments, each of which was performed in triplicate. In both panels, *indicates values that are significantly different (P < 0.01) from 50 mM KCl. c, CaMKII is necessary for ERK, but not Akt phosphorylation in response to KCl. Western blot analysis of equal amounts of protein derived from sympathetic neurons stimulated for 15 min with NGF (ng/ml) and/or KCl (mM) with or without 10 μM KN-62. Blots were probed with antibodies specific to tyrosine/threonine-phosphorylated ERKs (P-tyr/thr ERK), serine-phosphorylated Akt (P-ser Akt), or to total ERK or Akt protein. The ERK panels derive from reprobes of the same blot, and the Akt panels from reprobes of another.
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Figure 9: The role of CaMKII in survival mediated by NGF and/or KCl. a and b, CaMKII is necessary for KCl-mediated survival, but plays no role in the synergistic survival seen with NGF plus KCl. NGF-selected sympathetic neurons maintained in NGF and/or KCl for two days with or without 10 μM KN-62 and survival was then monitored by MTT assay (a) or TUNEL (b). In a, results represent the mean ± SD of one representative experiment performed in triplicate. Similar results were obtained in three separate experiments. In c, results represent the mean percentage of TUNEL-negative cells/total Hoechst-positive nuclei as determined in three separate experiments, each of which was performed in triplicate. In both panels, *indicates values that are significantly different (P < 0.01) from 50 mM KCl. c, CaMKII is necessary for ERK, but not Akt phosphorylation in response to KCl. Western blot analysis of equal amounts of protein derived from sympathetic neurons stimulated for 15 min with NGF (ng/ml) and/or KCl (mM) with or without 10 μM KN-62. Blots were probed with antibodies specific to tyrosine/threonine-phosphorylated ERKs (P-tyr/thr ERK), serine-phosphorylated Akt (P-ser Akt), or to total ERK or Akt protein. The ERK panels derive from reprobes of the same blot, and the Akt panels from reprobes of another.

Mentions: Together, these data indicate that Ras–PI3-kinase–Akt is one of the survival pathways induced by activation of neuronal L-type channels, and that this pathway is essential for the synergy between NGF and depolarization. A second, calcium-activated pathway that might also be involved in neuronal survival involves calcium/calmodulin-dependent protein kinase II (CaMKII; Hanson and Schulman 1992; Ghosh and Greenberg 1995). To determine the potential importance of this pathway for sympathetic neuron survival, neurons were maintained in various concentrations of NGF and/or KCl plus or minus 10 μM KN-62, a specific pharmacological blocker of CaMKII (Tokumitsu et al. 1990). MTT assays two days later revealed that 10 μM KN-62 dramatically reduced neuronal survival in 50 mM KCl, but had no effect on survival mediated by 10 ng/ml NGF or by 5 ng/ml NGF plus 10 mM KCl (Fig. 9 a). TUNEL assays confirmed this result: the number of TUNEL-negative cells was similar in NGF plus or minus KN-62, but a large decrease in TUNEL-negative cells was observed when KN-62 was added to 50 mM KCl (Fig. 9 b). Thus, in addition to a Ras–PI3-kinase–Akt pathway, calcium influx through L-type channels also mediates neuronal survival through a CaMKII-dependent pathway.


Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival.

Vaillant AR, Mazzoni I, Tudan C, Boudreau M, Kaplan DR, Miller FD - J. Cell Biol. (1999)

The role of CaMKII in survival mediated by NGF and/or KCl. a and b, CaMKII is necessary for KCl-mediated survival, but plays no role in the synergistic survival seen with NGF plus KCl. NGF-selected sympathetic neurons maintained in NGF and/or KCl for two days with or without 10 μM KN-62 and survival was then monitored by MTT assay (a) or TUNEL (b). In a, results represent the mean ± SD of one representative experiment performed in triplicate. Similar results were obtained in three separate experiments. In c, results represent the mean percentage of TUNEL-negative cells/total Hoechst-positive nuclei as determined in three separate experiments, each of which was performed in triplicate. In both panels, *indicates values that are significantly different (P < 0.01) from 50 mM KCl. c, CaMKII is necessary for ERK, but not Akt phosphorylation in response to KCl. Western blot analysis of equal amounts of protein derived from sympathetic neurons stimulated for 15 min with NGF (ng/ml) and/or KCl (mM) with or without 10 μM KN-62. Blots were probed with antibodies specific to tyrosine/threonine-phosphorylated ERKs (P-tyr/thr ERK), serine-phosphorylated Akt (P-ser Akt), or to total ERK or Akt protein. The ERK panels derive from reprobes of the same blot, and the Akt panels from reprobes of another.
© Copyright Policy
Related In: Results  -  Collection

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Figure 9: The role of CaMKII in survival mediated by NGF and/or KCl. a and b, CaMKII is necessary for KCl-mediated survival, but plays no role in the synergistic survival seen with NGF plus KCl. NGF-selected sympathetic neurons maintained in NGF and/or KCl for two days with or without 10 μM KN-62 and survival was then monitored by MTT assay (a) or TUNEL (b). In a, results represent the mean ± SD of one representative experiment performed in triplicate. Similar results were obtained in three separate experiments. In c, results represent the mean percentage of TUNEL-negative cells/total Hoechst-positive nuclei as determined in three separate experiments, each of which was performed in triplicate. In both panels, *indicates values that are significantly different (P < 0.01) from 50 mM KCl. c, CaMKII is necessary for ERK, but not Akt phosphorylation in response to KCl. Western blot analysis of equal amounts of protein derived from sympathetic neurons stimulated for 15 min with NGF (ng/ml) and/or KCl (mM) with or without 10 μM KN-62. Blots were probed with antibodies specific to tyrosine/threonine-phosphorylated ERKs (P-tyr/thr ERK), serine-phosphorylated Akt (P-ser Akt), or to total ERK or Akt protein. The ERK panels derive from reprobes of the same blot, and the Akt panels from reprobes of another.
Mentions: Together, these data indicate that Ras–PI3-kinase–Akt is one of the survival pathways induced by activation of neuronal L-type channels, and that this pathway is essential for the synergy between NGF and depolarization. A second, calcium-activated pathway that might also be involved in neuronal survival involves calcium/calmodulin-dependent protein kinase II (CaMKII; Hanson and Schulman 1992; Ghosh and Greenberg 1995). To determine the potential importance of this pathway for sympathetic neuron survival, neurons were maintained in various concentrations of NGF and/or KCl plus or minus 10 μM KN-62, a specific pharmacological blocker of CaMKII (Tokumitsu et al. 1990). MTT assays two days later revealed that 10 μM KN-62 dramatically reduced neuronal survival in 50 mM KCl, but had no effect on survival mediated by 10 ng/ml NGF or by 5 ng/ml NGF plus 10 mM KCl (Fig. 9 a). TUNEL assays confirmed this result: the number of TUNEL-negative cells was similar in NGF plus or minus KN-62, but a large decrease in TUNEL-negative cells was observed when KN-62 was added to 50 mM KCl (Fig. 9 b). Thus, in addition to a Ras–PI3-kinase–Akt pathway, calcium influx through L-type channels also mediates neuronal survival through a CaMKII-dependent pathway.

Bottom Line: This convergent PI3-kinase-Akt pathway was essential for synergistic survival.In contrast, inhibition of calcium/calmodulin-dependent protein kinase II revealed that, while this molecule was essential for depolarization-induced survival, it had no role in KCl- induced Akt phosphorylation, nor was it important for synergistic survival by NGF and KCl.This convergent regulation of Akt may provide a general mechanism for coordinating the effects of growth factors and neural activity on neuronal survival throughout the nervous system.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4.

ABSTRACT
In this report, we have examined the mechanisms whereby neurotrophins and neural activity coordinately regulate neuronal survival, focussing on sympathetic neurons, which require target-derived NGF and neural activity for survival during development. When sympathetic neurons were maintained in suboptimal concentrations of NGF, coincident depolarization with concentrations of KCl that on their own had no survival effect, synergistically enhanced survival. Biochemical analysis revealed that depolarization was sufficient to activate a Ras-phosphatidylinositol 3-kinase-Akt pathway (Ras-PI3-kinase-Akt), and function-blocking experiments using recombinant adenovirus indicated that this pathway was essential for approximately 50% of depolarization-mediated neuronal survival. At concentrations of NGF and KCl that promoted synergistic survival, these two stimuli converged to promote increased PI3-kinase-dependent Akt phosphorylation. This convergent PI3-kinase-Akt pathway was essential for synergistic survival. In contrast, inhibition of calcium/calmodulin-dependent protein kinase II revealed that, while this molecule was essential for depolarization-induced survival, it had no role in KCl- induced Akt phosphorylation, nor was it important for synergistic survival by NGF and KCl. Thus, NGF and depolarization together mediate survival of sympathetic neurons via intracellular convergence on a Ras-PI3-kinase-Akt pathway. This convergent regulation of Akt may provide a general mechanism for coordinating the effects of growth factors and neural activity on neuronal survival throughout the nervous system.

Show MeSH
Related in: MedlinePlus